Anopheles gambiae s.l (Diptera: Culicidae) seasonal abundance, abdominal status and parity rates in Metema-Armachiho lowland, Northwest Ethiopia.

Background Malaria is a life threating vector borne disease caused by different Plasmodium parasites. Metema and Armachiho are two of the top five malaria endemic areas among the districts of Amhara region in Ethiopia. Transmission pattern is seasonal and migrant laborers who visit these areas for employment in mechanized agriculture are highly affected. The aim of this study was to investigate seasonal abundance, abdominal status and parity rate of An.gambiae s.l in Metema-Armachiho lowlands, Northwest Ethiopia. Method A 1 year longitudinal entomological study was conducted in Metema-Armachiho lowlands from June 2016–May 2017. Mosquitoes were collected using CDC-light traps in indoor and outdoor sites for four consecutive days in each month. A total of eight standard battery operated CDC-light traps were used to collect mosquitoes. Female mosquitoes were classed as unfed, fed or gravid under a dissecting microscope. The ovaries of all unfed An.gambiae s.l mosquitoes were examined for evidence of parity. Data were entered and analyzed using SPSS-20 software. Chi-square test was applied to show significant difference between variables. P-value< 0.05 was taken as statistically significant. Results Of the total 1253 mosquitoes collected, 713 (552 female, 161 male) were culex and 540 (501 female, 39 male) were An.gambiae s.l. About 50.9% were collected in June–August 2016, 21.7% in September–November 2016, 12.0% in December 2016–February 2017 and 15.4% in March to May 2017. Of the total, 57.2 and 42.8% of the An.gambiae s.l were collected from indoor and outdoor sites respectively. Of the total females collected, 76.8% were unfed; of which 69.4% were parous. Significantly higher number of female An.gambiae s.l were collected in indoor and there was significant difference in abdominal status of An.gambiae s.l mosquitoes collected in different season (P < 0.05). Conclusions Highest number of An.gambiae s.l was observed from June–August followed by September–November. The parity rate of An.gambiae s.l was high and there was significant difference in abdominal status of An.gambiae s.l collected in different season.


Background
Malaria burden is high in poor and malnourished populations [1]. Labor migrants who travel to endemic areas, under five children and pregnant women are high risk groups for malaria infection [2][3][4]. Malaria transmission in Ethiopia is seasonal and affected by rainfall and altitude patterns [5][6][7]. Transmission period varies from a few weeks prior to the main rainy season (April-June) to more than a month following main rainy season (September-December) which coincides with the major harvesting and planting seasons [5,8,9]. Thus, it has a severe impact on Ethiopia's economy by cutting down productivity capacity [10].
Family Culicidae consists of two principal lineages that are recognized as subfamilies, the Anophelinae and Culicinae [11]. Of the 465 properly identified Anopheles mosquito species worldwide, 70 have the ability to transmit malaria parasites in humans [12]. An. arabiensis, which belongs to An.gambiae s.l, is the primary vector of malaria parasite in Ethiopia. However, An. pharoensis, An. nili and An. funestus are secondary malaria vectors [13][14][15]. A large decrease of the important endophilic-endophagic malaria vectors (An. gambiae sensu stricto and An. funestus) and persistence of the more exophilic-exophagic species (An. arabiensis) have been observed now a days [15,16].
Regular indoor residual spraying (IRS) and long lasting insecticide treated nets (LLITNs) are currently vector control intervention strategies in Metema and Armachiho [5,7]. Environmental management is also key intervention measure applied to control malaria in the study areas [13,17]. Modification of the environment has great effect on breeding of mosquitoes. It affects mosquito abundance by altering both quality and quantity of breeding sites [18].
Effectiveness of malaria prevention with LLITNs and IRS is inadequate due to emergence of insecticide resistance, outdoor and early evening/morning biting mosquitoes. New vector control tools are required and their efficiency should be improved. Therefore, studying An. gambiae s.l mosquito abundance, abdominal status and parity rate in Metema and Armachiho lowlands, which are among the hot spot areas of malaria in Ethiopia, is essential to design and apply complementary vector control tools that target outdoor and early morning/evening bitters to scale up malaria prevention and to reduce the burden of malaria in the study area.

Study area
The study was conducted in Metema-Armachiho lowlands, Northwest Ethiopia, considered as single survey domain, in three randomly selected villages; Kumer (13°00′N/36°15′E), Selam ber (13°45′N/36°24′E) and Abderafi (13°41′N/36°28′E). The altitude of Metema and West Armachiho Districts is 685 and 667 m above sea level respectively. The minimum annual temperature for both districts ranges from 22°C to 28°C. The daytime temperature is very high during March to May (reaches up to 43°C). These districts are hyper-endemic malarious areas and the weather condition is appropriate for mosquito vector breeding [19].

Study design and period
A 1 year longitudinal entomological study was conducted from June 2016 to May 2017 in Metema-Armachiho lowlands, Northwest Ethiopia. Entomological surveys were conducted in three villages (four houses from each village) using eight CDC-light traps in indoor and outdoor.

Mosquito collections
Mosquitoes were collected daily according to the WHO standard protocol using standard battery operated CDClight traps set overnight (18,00-06:00 h) [20,21]. A total of 12 houses, four houses from each village, were selected for the survey. Collection was made from 12 houses for four consecutive days in each month. A total of eight CDC-light traps were used to collect mosquitoes indoor and outdoor at 1-2 km distance from potential breeding sites. They were hanged 1.5 m above the ground. Indoor collection was performed in bedroom and outdoor collection was performed in verandas.

Anopheles gambiae s.l mosquito processing
All collected adult An. gambiae s.l mosquitoes were first anesthetized with ethyl ether for 30 min in an anesthetizing chamber. The sex and species of all collected mosquitoes were identified using standard keys [22][23][24] and labeled according to date and site of collection. Female mosquitoes were classed as unfed, fed or gravid under a dissecting microscope.

Dissection and examination of ovaries
The ovaries of all unfed An.gambiae s.l mosquitoes were examined for evidence of parity by observing whether they contained eggs or whether the tracheolar skeins surrounding the ovaries were coiled or uncoiled [20][21][22].

Data analysis and interpretation
The data was appropriately coded, entered, checked for completeness and analyzed using SPSS 20 software. Descriptive statistics, frequency and cross tabulations were used. The findings were presented in text, table and graph. Chi-square test was used for statistical analysis to show significant difference between variables. P-value< 0.05 was taken as statistically significant.

Indoor-outdoor distribution of mosquitoes across villages
In Kumer, approximately equal number of mosquitoes were collected in indoor and outdoor. In Abderafi, higher number of mosquitoes were collected in indoor than outdoor whereas, in Selam Ber there is a slight increase in outdoor. Significantly highest number (235) of mosquitoes were collected from Kumer (χ2 = 32.9; P < 0.05) (Fig. 1).

Discussion
This entomological study was the first time study carried out in Northwest Ethiopia to assess An. gambiae s.l. seasonal abundance, abdominal status and parity rate. Molecular analysis of An.gambiae s.l mosquitoes revealed that An. arabiensis is the only vector member of the complex in malaria endemic areas of Ethiopia [14,25,26]. Larval ecology indicates that An. gambiae s.l larvae develop in freshwater habitats that are small, temporary, clean and sun-exposed. The larva may breed in any available water, even in dirty and polluted habitats [27].
In this study, the highest number of An.gambiae s.l. were collected during rainy season of which 57.2% were from indoor sites and 42.8% were from outdoor sites  indicating the outdoor host seeking and resting behavior of An.gambiae s.l. Significantly higher number of male An.gambiae s.l were collected in outdoor and significantly higher number of female An. gambiae s.l were collected in indoor (P < 0.05). This indicates that only female mosquitoes seek blood for the development of their egg and male normally can survive by feeding on nectar juice. This is comparable with a previous study conducted in Sille, South Ethiopia which demonstrated that 54.2 and 45.8% of An.gambiae s.l were collected from indoor and outdoor sites respectively [28]. On the other hand, this finding is somewhat different from a previous study conducted in other part of Ethiopia, in which 44.4% of An.gambiae s.l were captured from indoor and 55.6% were collected from outdoor [29]. The reason for different proportions of indoor or outdoor biting might be due to human behaviour. In the study area (Metema and Armachiho district) migrant laborers often work outdoor and sleep in the open field exposing themselves for outdoor biters. This affects host seeking behaviors of An.gambiae s.l.
Of all female An.gambiae s.l mosquitoes examined for abdominal status, 76.8% were found unfed, 14.4% were found fed and, 8.8% were found gravid. The gravid An.gambiae s.l mosquitoes collected outdoor were either coming out to seek resting habitat or they were initially unfed indicating their outdoor resting and biting (partial exophillic-exophagic) behavior. There was significant difference in abdominal status of An.gambiae s.l mosquitoes collected in different seasons (P < 0.05).
Of all unfed female An.gambiae s.l mosquitoes examined, 69.4% were found parous and 30.6% were nulliparous. There was no significant difference in parity rate of An.gambiae s.l mosquitoes collected in different seasons (P > 0.05). This finding is comparable with previous study conducted in Sille, South Ethiopia with an overall parous rate of 73.2% [28] and higher than a study conducted in East Wellega Zone, West Ethiopia with an  overall An. arabiensis parous rate of 45% [14]. The reason for this difference might be due to difference in mosquito sampling season, mosquito sampling duration and host availability.

Conclusions
An.gambiae s.l abundance was highest in June-August followed by September-November. The parity rate of An.gambiae s.l was high and there was significant difference in abdominal status of An.gambiae s.l collected in different season. Supplementary vector control tools like larval source management and use of vapor phase repellents are required to tackle outdoor or early biting. In addition, there should be active surveillance including asymptomatic carriers.